Functional heterogeneity of Hcrt neurons Complex neuronal circuits regulate arousal, and deficiencies in these circuits are a highly prevalent cause for sleep disturbances and anxiety disorders. The multi-functional hypocretin/orexin (Hcrt) neurons regulate arousal-related behavioral states including sleep, wakefulness, feeding, emotions, stress and reward. However, how a presumably uniform Hcrt population regulates such diverse functions, and how alteration in these circuitries result in sleep and anxiety disorders is not clear. We hypothesize that Hcrt neurons are genetically, anatomically and functionally heterogenous, and that subpopulations of Hcrt neurons projecting to different brain regions regulate specific features of sleep and arousal. Here, we will use a combination of viral monosynaptic circuit tracing, two-photon live imaging of single synapse and neuronal activity, optogenetics, fiber photometry and behavioral experiments in zebrafish and mice to identify and elucidate the function of subpopulations of Hcrt neurons in regulating sleep. In aim 1, we will use retrogradely transported viral vectors and live imaging of single pre- and post-synaptic structure to characterize anatomically distinct subpopulations of Hcrt neurons, innervating the VTA, LC and TMN regions. The transparent zebrafish model enables anatomical and functional live visualization of the relatively simple, but conserved, Hcrt system (20-40 cells). In aim 2, fiber photometry recording in behaving mice and real-time two-photon imaging of neuronal activity in single cell resolution will be performed to elucidate the differential neuronal activity of subpopulations of Hcrt neurons in regulating sleep and arousal. In aim 3, optogenetic, synaptic silencing and genetically induced neuron ablation will be used to manipulate subpopulations of Hcrt neurons. We will monitor the effect on neuronal activity in post synaptic dopaminergic and histaminergic target neurons using genetically encoded calcium indicators. These neuronal activity recordings will be complemented with behavioral experiments in mice. The results are expected to challenge the generalized assumption that neurons that secrete a specific neuropeptide share similar identities and functions. The combined strength of the two vertebrate models will uncover the role of evolutionary conserved sub neuronal circuitries and may provide new therapeutic targets for the treatment of sleep disorders.